Development Of A Monoclonal Antibody Against SAMHD1Protein And Its Preliminary Application

SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11, is a kind ofnucleus protein which has deoxyribose nucleoside triphosphate hydrolase activity. SAMHD1canhinder early replication of human immunodeficiency virus type Ⅰ(HIV-1) in dendritic cells andother myeloid cells，but VPX coded by HIV-2and simian immunodeficiency virus（SIV）cancounteract this restrictions. The specific restrictions and antagonism is an important finding for thestudy of mechanism of virus invasion. The monoclonal antibody is an important tool for the studyof protein function. In this study, we prepared efficient secretion of anti-SAMHD1protein specificmonoclonal antibodies using prokaryotic expressed SAMHD1protein and conducted a preliminaryapplication.The SAMHD1gene amplified by RT-PCR was cloned into pcDNA3.1and pET30a.Throughthe optimization of prokaryotic expression condition, a recombinant protein of79kDa wasexpressed mostly in soluble form which was subsequently purified by Ni+-column. The expressionof both prokaryotic and eukaryotic recombinant proteins were confirmed by western blot.In this study，the4weeks-old BALB/c mice were intraperitoneally immunized with thepurified and prokaryotic expression protein of huSAMHD1. Then the spleen cells of theimmunized mouse were fused with SP2/0myeloma cells. In this study, we prepared a hybridomacell line3C3which could stably produce monoclonal antibodies whose antibody subtypes wasIgG2b/κ.The results of Western blot showed that3C3MAb could react with SAMHD1proteinswhich were expressed by pcDNA-huSAMHD1, and the endogenous expressed SAMHD1proteinsin293T cells and human THP-1cells. Meanwhile,3C3MAb could not react with SAMHD1proteins of monkeys or horses either expressed by eukaryotic expression vectors or the endogenousproteins in horse skin cells, donkey skin cells or rabbit kidney cells.This result indicated that theMAb3C3only recognize SAMHD1from human and had no cross-reactivity with SAMHD1fromother species, showing a good species specificity. The monoclonal antibody prepared in our studycan be used in immunofluorescence, western blot, ELISA and other immunological methods.The SAM and HD are two functional domains of SAMHD1. To identify that which domain isthe target of Mab3C3, we constructed the prokaryotic expression plasmid of pET-SAMHD1-SAMand pET-SAMHD1-HD. After transforming both of them into competent cells and inducingexpression by IPTG，two recombinant proteins were successfully expressed and their relativemolecular mass were26kDa and62kDa, respectively. The results of Western blot showed that 3C3MAb only reacted with the HD of SAMHD1, but not reacted with SAM of SAMHD1,indicating that the antigen epitope which3C3MAb recognizes is located in the HD domain ofSAMHD1.